J. Biol. Chem., Vol. 268, Issue 32, 24232-24241, Nov, 1993
Isolation, partial characterization, and molecular cloning of a human colon adenocarcinoma cell-surface glycoprotein recognized by the C215 mouse monoclonal antibody
P Bjork, U Jonsson, H Svedberg, K Larsson, P Lind, J Dillner, G Hedlund, M Dohlsten and T Kalland
Department of Immunology, Kabi Pharmacia AB, Lund, Sweden.
The monoclonal antibody C215 (IgG2a) was obtained by the immunization of
BALB/c mice with the human colon adenocarcinoma cell line COLO 205 and used
in the targeting of colorectal carcinomas. The partial characterization and
purification of the C215 target molecule from solubilized COLO 205
membranes indicated that it is an integral membrane glycoprotein of the
non-mucin type. The denatured antigen appeared as a major 40-kDa form in
Western blots after SDS- polyacrylamide gel electrophoresis and migrated as
a monomeric 36-kDa species after the reductive cleavage of intramolecular
disulfide bridges. Using a five-step procedure, the antigen was purified
4,300- fold from COLO 205 tumors raised in nude mice to a homogeneity of
95% when assessed by capillary electrophoresis. Removal of N-linked
carbohydrate by peptide:N-glycosidase treatment did not affect the
visualization of the purified antigen in immunoblots but resulted in a
faster migration in the SDS gels. The amino acid sequence was partially
determined. Seventeen contiguous NH2-terminal amino acids were identified
and coincided exactly with residues 82-98 of the GA733-2 protein cloned by
Szala et al. (Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewske,
Z., Koprowski, H., and Linnenback, A. J. (1990) Proc. Natl. Acad. Sci.
U.S.A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this
protein was used to prepare overlapping synthetic peptides that cover the
entire extracellular domain in order to identify the C215 epitope. A likely
epitope, close to the NH2 terminus and corresponding to the first distinct
hydrophilic stretch after the putative signal sequence, was identified in a
peptide enzyme- linked immunosorbent assay. Moreover, GA733-2 cDNA was used
for the cloning of the C215 protein from COLO 205 cells and the subsequent
transfection to K36.16 mouse T cell leukemia cells. The transfected cells
were C215 reactive in fluorescence-activated cell sorter analysis, and a 42
kDa band was visualized in Western blots under both non-reducing and
reducing conditions. Our findings indicate a close relationship between the
C215 antigen and other members of the GA-733 family, some of which are
currently being used as targets in clinical trials with monoclonal
antibodies. The mammalian expression system described here will enable
further studies into the biological role of this protein and the
construction of animal models in order to develop optimal therapeutic
strategies.
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